Molecular Characterization of the Human La Protein·Hepatitis B Virus RNA.B Interaction in Vitro

Abstract

The La protein was recently identified as a host factor potentially involved in the cytokine-induced post-transcriptional down-regulation of hepatitis B virus (HBV) RNA. The La binding site was mapped to a predicted stem-loop structure within a region shared by all HBV RNAs, and it was concluded that the La protein might be an HBV RNA-stabilizing factor. To characterize the RNA binding mediated by the different RNA recognition motifs (RRMs) of the human La protein, several La deletion mutants were produced and analyzed for HBV RNA binding ability. The data demonstrate that the first RRM is not required for binding, whereas the RNP-1 and RNP-2 consensus sequences of the RRM-2 and RRM-3 are separately required for binding, indicating a cooperative function of these two RRMs. Furthermore, the results suggest that multimeric La disassembles into monomeric La upon binding of HBV RNA.B. By gel retardation assay the affinity of the wild type human La⅐HBV RNA.B interaction was determined in the nanomolar range, comparable to the affinity determined for the mouse La⅐HBV RNA.B interaction. This study identified small regions within the human La protein mediating the binding of HBV RNA. Hence, these binding sites might represent targets for novel antiviral strategies based on the disruption of the human La⅐HBV RNA interaction, thereby leading to HBV RNA degradation.The human La protein is a 47-kDa phosphoprotein predominantly localized in the nucleus. It was first discovered as an autoantigen recognized by antibodies present in sera of patients suffering from systemic lupus erythematosus and Sjögren's syndrome (1, 2). The La protein is a member of a large group of RNA-binding proteins containing RNA recognition motifs (RRM) 1 (3-8) and is implicated in several steps of RNA metabolism. Among the different La proteins identified in a variety of organisms, the N-terminal part is highly conserved (9). La was shown to co-immunoprecipitate with a number of small RNA molecules (10). A role for La in the termination of RNA polymerase III transcription has been described. It was shown that La interacts with RNA polymerase III transcripts such as pre-tRNA by binding to a small stretch of uridines at the 3Ј-end common to these transcripts and might be necessary for proper processing of these precursors (11-17). In addition, La is known to interact with a variety of viral and other cellular . La is also suggested to be involved in the capindependent translation initiation of several viruses, including polio virus and hepatitis C virus (19,(27)(28)(29), and more recently evidence is growing that La stabilizes various RNAs, such as histone and hepatitis C and B virus RNA (22,23,25,30,31). At this time point it is not clear yet how La fulfills all of these different functions, however, assuming that this protein acts as a RNA chaperone, thereby stabilizing RNA structures, a function in these varied processes might be envisaged.The human La protein contains three RNA recognition motifs (RRM) involved in the binding of RNAs (9), although t...

Functional Characterization of the Interaction between Human La and Hepatitis B Virus RNA

“…In addition it has been shown that recombinant hLa interacts with in vitro transcribed HBV RNA.B (17). These data suggest an association of La and HBV RNA in living cells.…”

“…In contrast highly purified recombinant hLa binds HBV RNA.B with high affinity but with low specificity (17). It was concluded that accessory factors are required to specify the interaction between La and HBV RNA.B.…”

“…Expression, Purification, Phosphorylation, Dephosphorylation of Recombinant hLa Protein, and Preparation of Nuclear Extracts-Recombinant hLa protein was prepared as described previously (17). Phosphorylation of recombinant hLa was performed for 3 h at 37°C in 30-l reactions containing 1 g of hLa, 0.2 l of casein kinase II (New England Biolabs), 3 l of 10ϫ reaction buffer, and 6 l of 1 mM ATP.…”

“…While trying to evaluate the intracellular mechanism(s) responsible for the cytokine-mediated post-transcriptional destabilization of HBV RNA, the mouse La autoantigen (homologue to human La autoantigen) has been identified. The La protein interacts with a small cis-acting element located within the viral RNA between nt 1275 and 1291 (16,17). The tight temporal correlation between the cytokine-mediated down-regulation of HBV RNA and the cytokine-induced fragmentation of full-length La led to the assumption that full-length La stabilizes HBV RNA by interacting with the cis-acting element (18).…”

Nuclear Trafficking of La Protein Depends on a Newly Identified Nucleolar Localization Signal and the Ability to Bind RNA

“…Two conserved amino acid signatures within the RRMs, referred to as RNP-1 and RNP-2 (RNP consensus sequence), are essential for RNA binding (42). Recently, we have shown that the RNP-2 of RRM2 in hLa is required for binding of hepatitis B virus RNA (43), and it seems reasonable to assume that this motif is also required for the binding of cellular precursor transcripts. La interacts with RNA polymerase III products by binding the poly(U) stretch at the 3Ј-end common to this class of transcripts (44) or, in the case of the RNA polymerase II product U3 snoRNA, after partial processing exposing a poly(U) stretch (17).…”